Hello Everyone,
I am doing iTRAQ (8plex reagent) experiment to identify and quantify the relative proteomic changes between control and test set of an organism. After labeling of different iTRAQ reagents for control and test, I have done MS/MS analysis by Orbitrap.
Q.1-During my quantification, I have found much deviation(peak abundance) between replicates for the same peptide.
Q.2-Some peptides are present in one set of replicate and absent in another set. I am not to understand why the same biological replicate showing such error? Due to this, I am not able to conclude properly.
Q.3 I have used pierce high pH peptide fractionation method instead of the conventional HPLC method for fractionation. Here I have used 8 different fractions eluted from pierce high pH peptide fractionation column for MS/MS run. All the peptides are seen in first two fractions. Further fractions I don't see any peptides in LC-MS/MS run. Want to know why all peptides eluted in first two fractions?
Can anyone have such experience?
Does anyone have experience in such kind of problem?
Please reply.
Hi,
Although my most 'recent' experience is with 4plex iTRAQ on a MALDI-ToF/ToF, I'll try to address some of your questions nonetheless..
Re Q.1: what do you mean by deviation in peak abundance? Is that the abundance of the reporter ions in your MS/MS data, or in the abundance at MS1 (peptide) level? And replicates -in this context- are injections of the same sample? Or separate sample preparations?
Re Q.2: regardless of the type of replicates, mass spectrometers can have difficulty in selecting all the same peptide precursor ions due to the complexity of the sample they're offered. As a result, not always all of the same peptides will be selected for MS/MS and hence will not be identified in all runs/replicates.
Re Q.3: what percentage of organic solvent did you use to elute your peptides from the Pierce column? It seems you started off at too high concentration of organic solvent, so you could consider using lower percentages of organic solvents for elution.
Did you dry and redissolve your fractions after fractionation and prior to injection onto the LC-MS? Fractions containing high amounts of organic solvent are likely not to bind your column and therefore will elute in the void volume of your LC-MS run (te remain undetected by MS..).
Hope this helps..!
Hi,
just to add up -
Q1 - without observing the actual method, it will be difficult to trouble shoot your problem. however, it is known that isobaric labeling suffers considerably from co-elution. since you don't have good fractionation, this may well be a significant source of variability. However, there may be other issues - labeling efficiency, or differences between runs (if you have more samples than labels), column issues, sample preparation issues, protein quantification, etc.
Q2 - Again, we'll need accurate description of your experimental procedures. it may be genuine, or due to technical issues with sample preparation.
Q3 - did you optimize the elution conditions? what % ACN did you use for elution?
from my experience (in a big proteomics facility), 99% of the problems are due to faults in sample preparation. your best chance is to review your procedures with someone experienced in proteomics.
Good luck!
D.
Dear Eef Dirksen -
Q.1the abundance of the reporter ions in MS/MS data, Replicates in the context of different sample.
Q.3 Protocol for fractionation used as per mentiond link-
https://assets.thermofisher.com/TFS-Assets/LSG/manuals/84868_highph_rp_peptidefract_UG.pdf
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